Transmission electron microscopy conducted conventionally involves the use of film, video cameras or CCD (charge coupled device) detectors to detect high energy charged particles directly or indirectly through the agency of a intervening electron-sensitive scintillator screen which allows information to be collected on photon sensitive devices. Existing detection techniques have drawbacks.
Transmission electron microscopy has many applications. One important application is to the analysis of protein structure. Protein crystallography is the premier method to determine the 3D structures of large proteins, DNA or RNA/protein complexes, and viruses.
Cryo-Electron Microscopy (“cryo-EM”) could be used as a substitute technique for protein crystallography. An advantage of cryo-EM over protein crystallography is that it does not depend on growing large crystals, a very time consuming and, in many cases, impossible task. The main drawback, however, is the low resolution of the structures obtainable with conventional technology. For virus analysis, resolutions of about 7.4 Å have been achieved, and for large protein complexes, e.g., ribosome, resolutions of about 11.5 Å have been achieved. Despite these resolution limitations, cryo-EM has been employed successfully in time-resolved experiments to reveal the dynamic aspects of protein interaction.
Two principle factors limit the resolution of the structures characterized with cryo-EM. The first is difficulties with specimen stabilization and radiation damage. The second are difficulties with the collection and processing of the very large data sets required for statistical analysis. The problems with specimen stabilization and radiation damage have been largely corrected with electron microscopes that feature exceptional coherence and stability, and low temperature stages, and that hold specimens at either liquid nitrogen or liquid helium temperatures. Features have been developed that essentially remove chromatic aberration, for example.
The use of film to record images is problematic. While film provides excellent modulation transfer functions, especially in comparison with commercial CCD cameras, it requires several post-acquisition steps, including development and digitization, that are cumbersome and time-consuming. Even prior to the post acquisition, the use of film is cumbersome. The loading and unloading of film into a typical transmission electron microscope is a time-consuming procedure, and if the particular procedures are not followed exactly for a given microscope then the exposed film can be ruined or the unexposed film can be loaded improperly.
It has been estimated that to get a 10 Å (Angstrom) resolution structure of a large protein complex like the ribosome, it would be necessary to collect 100,000 images. We estimate that 3 Å resolution of a structure would require up to one million images with film. This renders present film detection techniques highly impractical, at the least.
CCD device detectors are also used for data detection in transmission electron microscopy. Commonly available CCD detectors for transmission electron microscopy have formats up to 4000×4000 pixels, although few, if any, commercial detectors deliver the full resolution of the device. The CCD detectors require the use of a phosphorescent scintillation screen to convert the electron image to a photonic image within a spectral range where the detector quantum efficiency is maximized. Unfortunately, with each electron event, a spot created within the scintillation screen is greater that the pixel pitch of the device. At 300 KeV, the full width at half maximum of the spot is around 30 μm; however, the full width at 1% is 200 μm. With a CCD pixel size of 15 μm×15 μm, the large spot size will effectively reduce a 2000×2000 pixel CCD to only 1000×1000 and a 4000×4000 pixel CCD to 2000×2000. To mitigate this problem, tapered fiber optics and/or demagnification lens optics can be used. However, large spatial distortions and non-uniformities, which are difficult to correct, are introduced by such optics. With demagnification lens-optics, the poor efficiency of the coupling dramatically reduces the number of photons reaching the CCD. CCD cameras for transmission electron microscopy can costs tens or hundreds of thousands of dollars.